A remarkable feature of the actin cytoskeleton is the coexistence within a single cell of diverse actin structures containing different sets of actin-binding proteins and exhibiting diverse morphologies, mechanical behavior, and turnover rates. Misregulation of the actin cytoskeleton and altered mechanical interactions with the extracellular matrix are associated with many forms of cancer and believed to contribute to differences in motility and morphology from normal cells. Recent studies have shown that mechanical stimuli can have a large impact on the motility and cytoskeletal morphology of cells, but mechanisms by which forces are converted into biochemical signals are only beginning to be uncovered. We propose to investigate the role that mechanical forces play in the regulation of the actin cytoskeleton. In particular, we focus on two proteins that stabilize or destabilize F-actin: tropomyosin and ADF/cofilin. These proteins affect the turnover of F-actin, which determines how quickly a cytoskeletal structure can be remodeled, and they may also play a role in determining network architecture and the particular complement of actin-binding proteins in the network by excluding some and including others. We hypothesize that the stress state of F-actin alters the binding of regulatory proteins to the sides of the filament. Rather than proteins having a single set of kinetic parameters that may depend only on the nucleotide state of the filament and on biochemical regulation of the proteins themselves, we hypothesize that protein binding may be promoted or inhibited by changes in tension on the filament. This idea is inspired in part by our recent finding that the branching complex Arp2/3 is more likely to bind to the outside of bent filaments than to straight filaments. In this R01 renewal application, we propose to test the idea that force on F-actin alters the binding and competition of ADF/cofilin and tropomyosin, both in vitro and in live cells, using a combination of unique force microscopy and fluorescence microscopy tools. The results of this study will provide the foundation for a new perspective on how cells may use mechanical stimuli to regulate actin cytoskeleton turnover and organization.